KR20220100491A - Pharmaceutical composition for preventing or treating gram-negative bacteria infectious diseases and antibacterial adjuvant for inhibiting resistance to antimicrobial agents - Google Patents

Abstract

The present invention relates to a composition for preventing or treating Gram-negative bacteria comprising a nanocomposite, in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated, and an antibacterial agent as active components. The nanocomposite, in which the nickel-doped zinc oxide and black phosphorus nanosheets are conjugated, acts specifically on Gram-negative bacteria containing the mcr-1 gene to neutralize positive charges on the surface of the bacteria and promote interaction with polymyxin to inhibit resistance to polymyxin. Also, the specific antibacterial activity against Gram-negative bacteria expressing the Mcr-1 gene is confirmed by increasing the antimicrobial sensitivity even with low-dose polymyxin treatment. Thus, the nanocomposite can be provided as an antibacterial adjuvant for inhibiting resistance to Gram-negative bacteria antimicrobial agents, and can be provided as a pharmaceutical composition for treating Gram-negative bacteria infections through combined treatment with a polymyxin-based antibacterial agent.

Description

Pharmaceutical composition for preventing or treating gram-negative bacteria infectious diseases and antibacterial adjuvant for inhibiting resistance to antimicrobial agents

The present invention relates to a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated; And it relates to a composition for preventing or treating a Gram-negative bacterial infectious disease containing an antibacterial agent as an active ingredient.

Infection with multidrug-resistant (MDR) Gram-negative bacteria results in a severe global mortality burden and increased mortality. In the medical environment, Gram-negative bacteria cause infections such as pneumonia, bloodstream infections, wound or surgical site infections, and meningitis, and patients in the intensive care unit (ICU) are particularly at high risk of these infections. The rapid resistance of Gram-negative bacteria to currently available antibiotics and their innate ability to withstand environmental pressures is a major concern for preventing or treating multidrug-resistant Gram-negative infections. Horizontal gene transfer is known as a major strategy for exchanging genetic material from one bacterium to another interacting bacterium to propagate drug resistance. However, due to the lack of effective antibacterial agents for controlling MDR-resistant bacteria, it was previously used to control MDR gram-negative bacteria infection. As a last resort, we are faced with the re-use of antibiotics.

Polymyxin, a last resort antibiotic, has been used in the treatment of MDR Gram-negative bacterial infections despite the risk of polymyxin resistance by EptA and ArnT, which transform lipid A with phosphoethanolamine (pEtN) residues and 4-amino-4-deoxy-1. It is used extensively. Fortunately, no strains naturally resistant to polymyxin were identified until 2015, but polymyxin resistance in which the colistin resistance-inducing gene (mcr-1) encoding Mcr-1, a type of protein included in the EptA family, can be transferred to a mobile form was confirmed, threatening the use of polymyxin as a last resort for infection with MDR gram-negative bacteria. Lipid A modification to lipopolysaccharide (LPS) on the bacterial surface using EptA reduces the negative charge on the surface of some Gram-negative bacteria. This imparts resistance to positively charged polymyxin through positive charge repulsion between the bacterial surface and polymyxin, thereby reducing polymyxin incorporation into the bacteria. Therefore, there is a need for the development of a new generation of antibiotics, inhibitors, alternative substances or therapeutic agents to control this resistance mechanism.

Republic of Korea Patent Publication No. 10-2017-0032689 (published on March 23, 2017)

The present invention provides a nanocomposite in which nickel-doped zinc oxide and black phosphorus are combined as an antimicrobial adjuvant to solve the problem of resistance to polymyxin antibacterial agent, which is a treatment for gram-negative bacteria, and to increase the sensitivity of the antibacterial agent to gram-negative bacteria, An object of the present invention is to provide an antibacterial pharmaceutical composition for Gram-negative bacteria that exhibit polymyxin resistance through combined treatment of a nanocomposite and an antibacterial agent.

The present invention relates to a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated; And it provides a pharmaceutical composition for preventing or treating Gram-negative bacterial infectious diseases containing an antibacterial agent as an active ingredient.

In addition, the present invention provides an antimicrobial adjuvant for inhibiting resistance to gram-negative bacteria antibacterial agents containing a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated as an active ingredient.

According to the present invention, the nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated acts specifically on Gram-negative bacteria containing the mcr-1 gene and exhibiting a positive charge on the surface, thereby neutralizing the charge on the surface of the bacteria. As the result of suppressing resistance to polymyxin by promoting polymyxin interaction and increasing antimicrobial sensitivity even with low-dose polymyxin treatment was confirmed, the nanocomposite may be provided as an antimicrobial adjuvant for suppressing resistance to antibacterial agents against gram-negative bacteria, , it can be provided as a pharmaceutical composition for the treatment of gram-negative bacterial infections through combination treatment with polymyxin-based antibacterial agents.

1 is a result of confirming the crystal phase, elemental composition, and atomic state of the nanocomposite, FIG. 1a is an XPS spectrum analysis result of ZO, NZO and NZB XRD patterns and NZB nanocomposite, FIG. 1b is a Zn 2p peak, FIG. 1c is Gaussian fitting curve of Ni 2p peak, Figure 1d is P 2p core peak.
2 is a result of confirming the shape and microstructure of the NZB sample, FIG. 2a is a TEM image, FIG. 2b is an HRTEM image, FIG. 2c is an element mapping image for zinc of the NZB sample, and FIG. 2d is an element for oxygen It is a mapping image, FIG. 2E is an elemental mapping image for Ni, and FIG. 2F is an elemental mapping image for P element.
Figure 3 is a result of confirming the change in the shape of NCCP16283, Figure 3a is a control, Figure 3b is a result of confirming the change in the shape of NCCP16283 treated with polymyxin B (PolB) (0.4 μg / mL), Figure 3c is NZB ( 125 μg/mL) is the result of confirming the change in the shape of NCCP16283 treated, and Figure 3d is the result of confirming the change in the shape of NCCP16283 treated with NZB (0.2 μg/mL) and PolB (62.5 μg/mL), the image is n = one of the representatives of 5.
Figure 4 is the result of confirming the surface charge of the nanomaterial and E. coli, Figure 4a is the result of confirming the zeta potential of BP, NZO and NZB, Figure 4b is the zeta potential of NZB in E. coli strains BW25113 and NCCP16283 that are not treated with NZB These are the measured results, and the BP, NZO and NZB concentrations are 125 μg/mL, and the result represents the average of n = 5 ± standard deviation (P <0.05).
Figure 5 is the result of confirming the cytotoxicity (WST analysis) by ZO, NZO and NZB exposure confirmed after treatment of NZB at a concentration of 10 and 25 μg / mL to HEK293 (human embryonic kidney) cells, Figure 5a is 24 hours After confirming the cytotoxicity, Figure 5b is the result of confirming the cytotoxicity after 48 hours, the error bar indicates the standard deviation (SD). P <0.05.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention found that the nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated contains the mcr-1 gene and acts specifically on Gram-negative bacteria that have a positive charge on the surface to neutralize the charge on the surface of the bacteria. - The present invention was completed by confirming the effect of suppressing resistance to polymyxin by enhancing the polymyxin interaction, and increasing the antimicrobial sensitivity even in a low-dose polymyxin treatment.

The present invention relates to a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated; And it can provide a pharmaceutical composition for preventing or treating Gram-negative bacterial infectious disease containing an antibacterial agent as an active ingredient.

The gram-negative bacteria may express the mcr-1 gene.

The mcr-1 gene may be to induce drug resistance to the polymyxin-based antibacterial agent exhibiting a positive charge by converting the charge on the surface of the Gram-negative bacteria to a positive charge.

The Gram-negative bacteria are Pseudomonas sp., Salmonella sp., Alcaligenes sp., Shigella sp., Pneumonia sp., Vibrio. sp.), may be selected from the group consisting of Neisseria sp. and Escherichia coli, but is not limited thereto.

The nanocomposite may be to neutralize the charge on the surface of Gram-negative bacteria exhibiting a positive charge by the mcr-1 gene, thereby suppressing the resistance of the antimicrobial agent and increasing the antimicrobial sensitivity.

According to an embodiment of the present invention, in the nanocomposite of the present invention, black phosphorus (BP), which is a negatively charged nanosheet, is expected to interact more effectively with gram-negative bacteria exhibiting a positive charge, including E. coli strains containing the mcr-1 gene. Accordingly, it is expected that the NZB binding to E. coli in which the Mcr-1 protein is expressed will increase the antibacterial effect of the antibiotic polymyxin B (PolB) exhibiting a positive charge, and to confirm this, E. coli strain BW25113, pQE60-mcr-1 BW25113, polymyxin-resistant strain (NCCP), and NDM-1 MDR strain together with a checker board analysis between PolB and NZB was performed to confirm the drug combination effect of NZB on polymyxin B.

As a result, as shown in the FICI values in Table 2, NZB vs. PolB showed the maximum synergistic effect (FICI _NZB/PolB = 0.175-0.225) against mcr-1 gene-expressing E. coli, but it was It was confirmed that the above effect did not appear for this (FICI _NZB/PolB = 2).

From the above results, it was confirmed that the antimicrobial activity enhancing effect of NZB on PolB was specific to Mcr-1 expressing E. coli.

The antibacterial agent may be one or two or more selected from the group consisting of polymyxin-based antibacterial agents consisting of polymyxin B and colistin.

The nanocomposite may be contained in an amount of 0.652 to 2.5 parts by weight based on 100 parts by weight of the total pharmaceutical composition.

The antimicrobial agent may be contained in an amount of 0.08 to 0.64 parts by weight based on 100 parts by weight of the total pharmaceutical composition.

The gram-negative bacterial infectious disease may be selected from the group consisting of skin infections, pneumonia, sepsis, enteritis, Crohn's disease, ulcerative colitis, endocarditis, meningitis, otitis externa, otitis media, osteomyelitis, urinary tract infection, respiratory tract infection, and wound infection.

In one embodiment of the present invention, the nickel-doped zinc oxide and black phosphorus nanosheets are combined nanocomposite; And the pharmaceutical composition for preventing or treating Gram-negative bacterial infectious disease containing an antibacterial agent as an active ingredient is an injection, granule, powder, tablet, pill, capsule, suppository, gel, suspension, emulsion, drop or solution according to a conventional method. Any one formulation selected from the group consisting of can be used.

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent, diluent commonly used in the preparation of pharmaceutical compositions. , may further include one or more additives selected from the group consisting of dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil can be used, and solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and these solid preparations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered to the subject.

Preferred dosages of the nanocomposite and the antimicrobial agent may vary depending on the condition and weight of the subject, the type and extent of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

The present invention can provide an antimicrobial adjuvant for suppressing resistance to gram-negative bacteria antibacterial agents containing a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated as an active ingredient.

The gram-negative bacteria may contain an mcr-1 gene, and may exhibit a positive charge on the bacterial surface by the Mcr-1 protein expressed by the mcr-1 gene.

The antibacterial agent may be one or two or more selected from the group consisting of polymyxin-based antibacterial agents consisting of polymyxin B and colistin.

The Gram-negative bacteria are Pseudomonas sp., Salmonella sp., Alcaligenes sp., Shigella sp., Pneumonia sp., Vibrio. sp.), may be selected from the group consisting of Neisseria sp. and Escherichia coli.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Preparation of Mcr-1 protein overexpression plasmid

To prepare the Mcr-1 protein overexpression plasmid, the DNA sequence of the Mcr-1 protein encoding gene was obtained from the NCBI protein database (GenBank ID: ASK04346.1), and the program of GenSmartTM Codon Optimization (GenScript, NJ, USA) was utilized. to optimize codons for protein expression. The optimized DNA sequence (Table S2) was synthesized and subcloned into pQE60 (Qiagen, CA, USA) linearized with BamHI/BglII. The subcloned plasmid DNA was finally confirmed by colony PCR and DNA sequencing analysis using primers pQE30-F and -R as primers. The obtained Mcr-1 protein overexpression plasmid was transformed into BW25113 strain for further experiments.

2. X-ray diffraction (XRD) confirmation

An X-ray diffractometer (D8 Advance, Billerica, Bruker, MA, USA, DAVINCI design) equipped with a nickel-filtered Cu Kα radiation source (λ = 1.5406 A) was used and to confirm the X-ray diffraction (XRD) pattern, 2θ ZO, NZO and NZB samples in the range 5 - 80 ° were prepared. The microstructure of a representative NZB sample was evaluated by transmission electron microscopy (TEM; Bruker Nano GmbH), in which a carbon-coated 300 mesh Cu grid was used. In addition, the binding energy and chemical state of the element in the representative NZB sample was confirmed using the Axis Supra scanning X-ray photoelectron spectroscopy (XPS) microprobe surface analysis system (Kratos Analytical, Manchester, UK) in the binding energy range of 200 to 200. , 1,200 eV. The C 1s peak position at 284.5 eV was used as the binding energy reference.

3. Zeta Potential Measurement

The zeta potential (millivolt, mV) is determined from the electrophoretic mobility μ at 25 °C in 1 mM NaCl using the Smoluchowski equation ζ = μη/ε. where η is the medium viscosity and ε is the medium dielectric constant of BP. NZO and NZB or E. coli suspensions in DW were measured with a Nanopartica SZ-100 (Horiba Scientific) according to the manufacturer's instructions. BW25113 and NCCP16283 E. coli strains were cultured overnight with or without a minimal inhibitory concentration (MIC) treatment of NZB (125 μg/mL) and diluted to OD ₆₀₀ = 0.5. Thereafter, the cell culture precipitate was obtained by centrifugation at 12,000 rpm for 2 minutes, washed three times with phosphate buffered saline (PBS), and resuspended in 1 mL of DW to measure the zeta potential. The zeta potential values shown for each sample are the mean of at least 5 measurements ± standard deviation (P < 0.05).

4. Bacterial growth conditions, strains used and plasmids

The strains and plasmids used are shown in Table 1. The strains were cultured on Luria-Bertani (LB) medium or agar plates with or without antibiotics. The Keio (E. coli K-12 in-frame, single-gene knockout mutants) collection was used after verifying the deletion of the target gene by performing colony PCR using k1 and gene-specific primers. -Taq HS (Toyobo, Japan) dye was mixed and used.

strains and plasmids Information BW25113 wild-type Keio-arnT arnT mutant Keio-eptA eptA mutant NCCP16283 E. coli isolate, mcr-1 NCCP16284 E. coli isolate, mcr-1 BAA-2471 E. coli isolate, NDM-1, β-lactamase-1 BAA-2340 E. coli isolate, NDM-1, β-lactamase-1 KS7000 ¹ BW25113, pQE60 KS8000 ² BW25113, pQE60-mcr-1 NCCP16285 Klebsiella isolate, mcr-1 ATCC19606 Acinetobacter baumannii ATCC27853 Pseudomonas aeruginosa pQE60 pQE60 plasmid pQE60-mcr-1 mcr-1 containing pQE60 plasmid

* KS7000 ¹ and KS8000 ² contain pQE60 or pQE60-mcr-1 plasmids in BW25113 strain.

* Abbreviations: PolB, polymyxin B; NZB, nickel doped zinc oxide with black phosphorus; FICI, fraction inhibitory concentration index/index; NDM-1, New Delhi Metallo-1; β-lactamase-1, β-lactamase-1; mcr-1: colistin resistance induction gene.

5. Expression analysis of Mcr-1 protein

After the expression of Mcr-1 protein from the pQE60-mcr-1 plasmid, expression of the protein extract was confirmed by performing SDS-PAGE gel electrophoresis and Western blotting. First, to confirm Mcr-1 protein expression, the protein extract was electrophoresed on a 12% Mini-PROTEAN Stain-free TGX Precast gel (Bio-Rad, CA, USA), followed by histidine tag (His-tag, Sino Biological, PA). , USA) and Clarity™ Western ECL substrate (Bio-Rad) was used to perform Western blotting for histidine-tagged Mcr-1 protein. Image analysis was performed using ChemiDocMP (Bio-Rad) and Image Lab (Ver.6.1.0, Bio-Rad), with one of the representatives of n = 3 indicated.

6. Preparation of Gram-negative bacteria for testing

The bacteria used to investigate the morphological characteristics of bacteria by antibacterial activity and antibacterial action were prepared as follows. First, Gram-negative bacterial colonies were plated on LB agar plates with or without antibiotics and resuspended in DW using a Sensititre ^™ Nephelometer (Thermo Fisher Scientific, MA, USA) to an optical density of 0.5 McFarland turbidity standard. Individual bacterial cultures were diluted 1,000-fold and inoculated in Sensititre ^™ cation-conditioned Mueller-Hinton medium.

7. Confirmation of antibacterial activity

The antibacterial samples were divided into 4 groups and prepared in the indicated concentration range as follows together with the control group. (1) NFW (untreated control), (2) NZB (0-250 μg/mL), (3) PolB (Polymyxin B, Sigma-Aldrich, 0-3.2 μg/mL) and (4) NZB (0- 250 μg/mL)/PolB (0-3.2 μg/mL) mixture.

In order to confirm the antibacterial activity of the antibiotic, prepared Gram-negative bacteria (section 2.4.5.) were dispensed into 96-well plates containing control, PolB, NZB and NZB PolB, and cultured at 37° C. for 16 hours without shaking. Minimum inhibitory concentration (MIC) and synergy as fractional inhibitory concentration index/index (FICI) were determined using checker board analysis [the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections, Clin Infect Dis . 40 (2005) 1333-1341.] was interpreted as described. At least three replicates were performed to evaluate the synergy between NZB and PolB.

8. Morphological Characteristics of Bacteria

In order to confirm the morphological change according to the material treatment of the bacteria, checker board analysis was performed. Four samples from the control, PolB, NZB and sub-MIC of NZB/PolB were centrifuged at 10,000×g for 1 min. The resulting bacterial culture precipitate was resuspended in 500 μL PBS containing 2% formaldehyde and 1% glutaraldehyde and centrifuged for 5 minutes. The final bacterial culture precipitate was washed twice and resuspended in 1 mL DW. A 5 μL aliquot from the suspension was collected and deposited on a silicon wafer (5 × 5 mm, Namgang Hi-Tech Co., Ltd., Seongnam, Korea) and dried at room temperature. The dried wafers were analyzed using a VEGA3, a versatile tungsten thermionic emission scanning electron microscope (SEM) system (TESCAN, Fuveau, France) according to the manufacturer's protocol.

9. Cytotoxicity analysis and morphological change confirmation

HEK293 cells from the American Type Culture Collection (Manassas, VA, USA) were maintained in RPMI1640 with 10% fetal bovine serum at 37°C in 5% CO ₂ . Cell viability was confirmed by colorimetric WST analysis (Ez-Cytox; DoGenBio, Seoul, Korea). Cells were seeded in 96-well plates at a density of 5,000 cells per well and incubated for 24 hours.

Thereafter, the cells were further cultured in 0.1% dimethyl sulfoxide in the presence of a NZB sample at a concentration of 10-200 μg/mL for 24 or 48 hours, followed by the addition of WST reagent (1/10 of the medium volume) to form the cells. The amount of formazan dye was checked for absorbance at 450 nm using a spectrophotometer microplate reader (BMG LABTECH GmbH, Ortenber, Germany). The morphology of HCT-15 cells treated with the sample was imaged using a phase contrast microscope (Leica DM IL LED; Leica, Wetzlar, Germany).

<Example 1> Synthesis of nanocomposite

1. Synthesis of Ni-Doped ZnO Nanoparticles (NZO)

NZO with 5 atomic percent (at %) ZnO-based NPs (with respect to Zn ²⁺ ) was found in a previous study [B. Mehrad, NM Clark, GG Zhanel, JP Lynch, Chest. 147 (2015) 1413-1421.] was synthesized as described.

2. Synthesis of BP nanosheets and Ni-doped ZnO-BP (NZB) nanocomposites

2-1. Synthesis of black phosphorus (BP) nanosheets

A previously reported method [Horizontal gene transfer mediated bacterial antibiotic resistance, Front. Microbiol. 10 (2019) 1933.] was used to synthesize BP nanosheets. In the synthesis process, an aqueous exfoliation process through ultrasonication was performed. First, 2.0 g of NaOH was added to 60 mL of N-methyl-2-pyrrolidone (NMP) solvent for 5 minutes under water bath sonication. Then, after centrifugation, the supernatant was collected. Then bulk BP crystals (25 mg) were added to a saturated NaOH solution containing NMP. The resulting suspension was sonicated for 8 hours using an ice bath sonicator maintained at a temperature below 20°C. Thereafter, the unexfoliated BP crystals were separated by centrifugation at 2,000 rpm for 15 minutes. The supernatant was subsequently collected and centrifuged again at 13,000 rpm for 10 min. Finally, the BP nanosheets collected in the centrifugation process were dispersed in water and stored at 4 °C for further use.

2-2. NZB Nanocomposite Synthesis

5 μL of the prepared BP nanosheets (2 mg/mL) was added to 40 mL of deionized water (DW) in which 100 mg of synthesized NZO was dissolved, and stirring was continued for 10 minutes. Thereafter, the mixture was sonicated for 10 minutes and then the mixture was stored for 6 hours with continuous stirring, after which the samples were collected by centrifugation and vacuum dried at 60° C. for 6 hours.

<Example 2> Confirmation of nanocomposite properties

1. Check the phase configuration

The NZO and NZB samples prepared in Example 1 and the crystalline phases of ZO were confirmed using XRD.

As a result, the XRD reflection peaks of ZO, NZO and NZB synthesized as shown in FIG. 1a were consistent with the hexagonal ZO (h-ZO) structure (JCPDS 36-1451). However, some additional peaks in the NZB sample were identified at around 16.78° and 52.38°. The peak can be attributed to the (020) and (060) lattice planes of BP.

From the above results, as it was confirmed that the BP nanosheets exfoliated without impurities after liquid sonication were still in the orthorhombic system, it was confirmed that the NZB nanocomposite was successfully formed.

2. XPS (X-ray photoelectron spectroscopy) confirmation

The elemental composition and valence state of a representative NZB sample were confirmed by XPS.

The binding energy signals of Zn 2p (Fig. 1b) and Ni 2p (Fig. 1c) were confirmed as in Figs. A peak was detected, which was assigned to the binding energy of the Zn 2p ^3/2 and Zn 2p ^1/2 states, respectively. Also, the calculated difference in binding energy between Zn 2p ^3/2 and Zn 2p ^1/2 was ~23.1 eV, confirming the valence state of Zn ²⁺ zinc.

1c is a Gaussian-fitting binding energy curve of Ni 2p, and the two main peaks were concentrated at about 856.2 and 873.5 eV, respectively, indicating the binding energies of the Ni 2p ^3/2 and Ni 2p ^1/2 states. Similarly, it was confirmed that the calculated energy difference between the Ni 2p ^{3/2 and} Ni 2p ^1/2 states was 17.3 eV, which showed a significant difference from NiO (18.4 eV), so that Ni present in NZB was different from NiO. .

In addition, the binding energy of the P 2p peak identified in FIG. 1d was confirmed to be consistent with the data of other reports previously published.

The successful formation of the NZB nanocomposite was confirmed from the XPS and XRD results.

3. Confirmation of morphology and microstructure of nanocomposites

The morphology and microstructure of a representative NZB was confirmed as shown in FIG. 2 .

Fig. 2a is a TEM image of NZB nanoparticles in which NZO nanoparticles are distributed on BP nanosheets, Fig. 2b is a high-resolution TEM (HRTEM) image of NZB, and the inset of Fig. 2b is an enlarged version in the indicated area of Fig. 2b, interplanar. A distinct grid with a distance of 0.25 corresponds to the (101) plane of the ZO. In addition, it was confirmed that Zn, O, Ni, and P elements exhibit good distribution, respectively, as shown in Figs. 2c, 2d, 2e, and 2f, which are elemental mapping images of representative NZB samples.

From the TEM and HRTEM images and elemental mapping results, it was confirmed that the NZB nanocomposite was successfully formed.

<Example 3> Confirmation of antibacterial activity

1. Confirmation of antibacterial activity of NZB against E. coli strains

First, the antibacterial activity was confirmed as the MIC of polymyxin B (PolB) against the standard, NCCP-resistant strains and NDM-1 MDR E. coli strains.

As a result, as shown in Table 2, the MIC value for BW25113 was confirmed to be 0.8 μg/mL in the condition in which the E. coli strain containing the control vector and NDM-1 was present or not, E. coli containing the used NDM-1 It was confirmed that the strain was still susceptible to PolB.

On the other hand, the NCCP-resistant strain containing the mcr-1 gene as a mobile element showed 4-fold higher resistance to PolB (MIC = 3.2 μg/mL) compared to the BW25113 strain. Since these resistant strains may have multiple resistance genes, it is difficult to determine the effect on antibiotic susceptibility. Accordingly, an expression vector capable of expressing only Mcr-1 protein was codon-optimized to maximize expression in Escherichia coli, followed by synthesizing the DNA sequence for the Mcr-1 protein coding region with a C-terminal 6 × histidine tag. Plasmids pQE60 and pQE60-mcr-1 were constructed.

The constructed plasmid was transformed into E. coli strain BW25113 and used to evaluate the effect of Mcr-1 protein on PolB sensitivity. Mcr-1 was expressed in E. coli using isopropyl β-d-1-thiogalactopyranoside, and as a result of Western blot analysis using an antibody against 6 × histidine tag, it was confirmed that Mcr-1 protein was properly expressed.

In addition, as a result of further MIC analysis of the strain, it was found that Mcr-1 protein overexpression increases resistance to PolB (MIC = 3.2 μg/mL) in the NCCP-resistant strain. From the above results, it was confirmed that the PolB resistance of the NCCP-resistant strain originated from Mcr-1 protein expression, and the role of Mcr-1 in multiple treatments can be evaluated through this system.

On the other hand, as a result of further confirming the antimicrobial activity of the nanocomposite against E. coli by performing MIC analysis, the MIC values of BP, NZO and NZB for all Gram-negative bacteria used in the experiment were > 500, 200 and 200, respectively, as shown in Table 2 μg/mL.

From the above results, it was confirmed that NZB exhibits antibacterial activity, but it was not possible to distinguish the strain characteristics, and it was confirmed that the antibacterial activity of NZB was indicated by NZO, not BP.

strain Monotherapy Synergistic combination MIC (μg/mL) MIC (μg/mL) Maximum FICI _NZB/PolB PolB NZB PolB Fold change to Monotherapy NZB Fold change to Monotherapy BW25113 0.8 250 0.8 One 250 One 2 NCCP16283 3.2 250 0.4 8 25 10 0.225 NCCP16284 3.2 250 0.4 8 25 10 0.225 BAA-2471 0.8 250 0.4 2 125 2 One BAA-2340 0.8 250 0.4 2 125 2 One KS7000 ^One 0.8 250 0.4 2 125 2 One KS8000 ² 6.4 250 0.8 8 12.5 20 0.175

* KS7000 ¹ and KS8000 ² contain pQE60 or pQE60-mcr-1 plasmids in BW25113 strain.

* Abbreviations: PolB, polymyxin B; NZB, nickel doped zinc oxide with black phosphorus; FICI, fraction inhibitory concentration index/index.

2. Confirmation of synergy between NZB and PolB

BP, which is a negatively charged nanosheet, is expected to interact more effectively with positively charged cells, including E. coli containing mcr-1. Therefore, NZB binding to mcr-1 expressing E. coli is positively charged, antibiotic PolB It was expected to increase the synergism with

To confirm this, checker board analysis between PolB and NZB was performed with E. coli strain BW25113, BW25113 of pQE60-mcr-1, NCCP-resistant strain, and NDM-1 MDR strain to confirm the synergistic effect of NZB.

Referring to the FICI values in Table 2, NZB versus PolB showed the greatest synergistic effect (FICI _NZB/PolB = 0.175-0.225) against the E. coli strain containing the mcr-1 gene, and did not appear for other strains including NDM-1. It was confirmed that not (FICI _NZB/PolB = 2).

From the above results, it was confirmed that the enhancement of the efficacy of NZB for PolB was specific to the E. coli strain expressing the Mcr-1 protein.

Polymyxin resistance is known to arise mainly from the structural remodeling of lipid A anchored to the bacterial surface, and ArnT and EptA modifications were observed in Mcr-1 protein-expressing strains. Accordingly, to determine whether inhibition of ArnT or EptA action is required for NZB synergy to PolB, whole mutants of arnT or eptA (Keio collections [Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants] : The Keio collection, Mol. Syst. Biol. 2 (2006) 2006.0008.]) was used to perform checker board analysis of NZB with PolB.

As a result, it was confirmed that the MICs of PolB and NZB against keio-arnT and -eptA were the same as those of BW25113 (MIC = 0.8 μg / mL), and antibacterial efficacy similar to that of NCCP16283 against keio-arnT and -eptA strains was confirmed. could be seen to appear.

From the above results, it was confirmed that suppression of ArnT or EptA expression was suitable for inducing a synergistic action of NZB on polymyxin B in E. coli.

<Example 4> Confirmation of morphological characteristics of bacteria

To confirm the antibacterial properties of PolB, NZO, NZB and NZB/PolB combination treatment, morphological analysis of bacteria (NCCP16283: resistant strain containing mcr-1) was performed under various treatment conditions using SEM.

As a result, the untreated E. coli as shown in FIG. 3A showed a typical bacterial shape with a smooth surface and a normal length. On the other hand, cell membrane disruption was confirmed in the bacteria treated with PolB, NZB and NZB/PolB as shown in FIGS. 3b, 3c and 3d. In particular, in NCCP16283, it was confirmed that the morphology of NZB and NZB/PolB-treated bacteria was significantly different from that of normal bacteria compared to PolB-treated.

From the above results, it was confirmed that NZB exhibits a different mechanism of action in bacterial killing, and as shown in Table 1, NZB exhibits a synergistic effect on PolB to kill more multidrug-resistant bacterial populations.

<Example 5> Confirmation of action mechanism of NZB (neutralizing charge on bacterial LPS)

Escherichia coli is generally known to exhibit a negative charge on the surface due to the negatively charged lipid A in the LPS component. However, Mcr-1 protein modification modifies ArnT and EptA, resulting in modification of the overall lipid A structure, thereby changing the surface charge of E. coli from negative to positive. Since NZB is a negatively charged nanocomposite, it was expected that charge neutralization between NZB and E. coli with a surface modified including the mcr-1 gene would make bacteria susceptible to attack by positively charged polymyxin B (PolB).

To confirm this hypothesis, the zeta potentials of BP, NZO and NZB samples were measured.

As a result, referring to FIG. 4a, the values of BP, NZO and NZB were -30.8, +11.2 and -41.2 mV, respectively. It was confirmed that the BP representing the negative charge in NZB was increased compared to the BP itself due to the integration of NZO, although NZO exhibited a positive charge. This increase in negative charge was confirmed in a previous report in which Li ⁺ incorporation increased the negative charge of BP [Negatively charged 2D black phosphorus for highly efficient covalent functionalization, Mater. Chem. Front. 2 (2018) 1700-1706.].

In addition, it was further confirmed whether the surface charge of E. coli strains BW25113 and NCCP16283 was changed by NZB treatment with a sub-MIC concentration (125 μg/mL).

As a result, as shown in FIG. 4b, the zeta potentials of the combination of BW25113 and NCCP16283 with NZB were confirmed to be -56.9 and -73.7 mV, respectively. On the other hand, the zeta potentials of BW25113 and NCCP16283 without NZB were -63.9 and -51.4 mV, respectively.

From the above results, it was confirmed that Gram-negative bacteria having more negative charges on the surface showed a relatively low interaction effect with NZB showing negative charges. Therefore, it was confirmed that Gram-negative bacteria modified by Mcr-1 protein reduced negative charge on the surface and interacted more with NZB to induce modification on the bacterial surface as shown in FIG. 3d. It was confirmed that it easily penetrates and induces a synergistic effect of PolB.

<Example 6> Cytotoxicity of NZB

To confirm the in vitro cytotoxicity of NZB, WST analysis was performed on HEK293 cells (human embryonic kidney cells) treated with various concentrations of NZB. To determine the lowest concentration for a synergistic effect between NZB and PolB with FICI _NZB/PolB = 0.175-0.225, additional checker board analyzes were performed using a range of NZB and PolB.

As a result, it was confirmed that cytotoxicity did not appear at the synergistic concentration of 12.5 μg/mL as shown in FIG. 5 .

From the above results, it was confirmed that the synergistic concentration of PolB and NZB nanocomposite was biocompatible and effective in the treatment of polymyxin-resistant bacterial infection.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are conjugated; And a pharmaceutical composition for preventing or treating Gram-negative bacterial infectious disease containing an antibacterial agent as an active ingredient. The pharmaceutical composition for preventing or treating Gram-negative infectious diseases according to claim 1, wherein the Gram-negative bacteria contain the mcr-1 gene and express Mcr-1 protein. The method according to claim 1, wherein the Gram-negative bacteria are Pseudomonas sp., Salmonella sp., Alcaligenes sp., Shigella sp., Pneumonia sp., Gram-negative bacterial infectious disease prevention or treatment pharmaceutical composition, characterized in that selected from the group consisting of Vibrio sp., Neisseria sp. and Escherichia coli. The gram-negative bacterial infectious disease according to claim 1, wherein the nanocomposite neutralizes the charge on the surface of the gram-negative bacteria showing a positive charge by the protein expressed by the mcr-1 gene, thereby suppressing the resistance to the antibacterial agent and increasing the antibacterial agent sensitivity. A pharmaceutical composition for prophylaxis or treatment. The pharmaceutical composition for preventing or treating Gram-negative bacterial infectious diseases according to claim 1, wherein the antibacterial agent is one or more selected from the group consisting of polymyxin-based antibacterial agents consisting of polymyxin B and colistin. The pharmaceutical composition for preventing or treating Gram-negative bacterial infectious diseases according to claim 1, wherein the nanocomposite is contained in an amount of 0.625 to 2.5 parts by weight based on 100 parts by weight of the total pharmaceutical composition. The pharmaceutical composition according to claim 1, wherein the antibacterial agent is contained in an amount of 0.08 to 0.64 parts by weight based on 100 parts by weight of the total pharmaceutical composition. The method according to claim 1, wherein the gram-negative bacterial infectious disease is selected from the group consisting of skin infection, pneumonia, sepsis, enteritis, Crohn's disease, ulcerative colitis, endocarditis, meningitis, otitis externa, otitis media, osteomyelitis, urinary tract infection, airway infection and wound infection. Gram-negative bacterial infectious disease prevention or treatment pharmaceutical composition, characterized in that. An antimicrobial adjuvant for suppressing resistance to gram-negative bacteria antibacterial agents containing a nanocomposite in which nickel-doped zinc oxide and black phosphorus nanosheets are combined as an active ingredient. The antimicrobial adjuvant for suppressing antibiotic resistance of Gram-negative bacteria according to claim 9, wherein the Gram-negative bacteria contain a mcr-1 gene, and exhibit a positive charge on the bacterial surface by the Mcr-1 protein expressed by the mcr-1 gene. The antimicrobial adjuvant for inhibiting antibiotic resistance of Gram-negative bacteria according to claim 9, wherein the antibacterial agent is one or more selected from the group consisting of polymyxin-based antibacterial agents consisting of polymyxin B and colistin. The method according to claim 9, wherein the Gram-negative bacteria are Pseudomonas sp., Salmonella sp., Alcaligenes sp., Shigella sp., Pneumonia sp., Antibacterial adjuvant for inhibiting antibiotic resistance of Gram-negative bacteria, characterized in that selected from the group consisting of Vibrio sp., Neisseria sp. and Escherichia coli.

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